Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Weiss, Project 1) The theme of this Program Project application is to understand the signal transduction pathways mediating cardioprotection using a multidisciplinary approach that combines biophysics, physiology, proteomics and genetics. Project 1 focuses on a central tenet of this theme, that cardioprotective signaling converges on protection of mitohchondria by preventing the mitochondrial permeability transition (MPT). Project 1 will characterize the role of two distinct components of MPT to predispose mitochondria to injury during anoxia/reoxygenation. The MPT priming component is most relevant to the anoxic, or ischemic, period and primes the mitochondria to undergo MPT during reperfusion. The MPT priming component manifests as progressive MPT-independent inner mitochondrial membrane (IMM) proton leak, matrix condensation and remodeling, and cytochrome c mobilization and release and is promoted by accumulation of long chain fatty acids (FA) and reactive oxygen species (ROS). The MPT trigger component is most relevant to the reoxygenation, or reperfusion, phase. Whether MPT occurs during reperfusion is determined by the interplay between MPT inducers and inhibitors present during rexoygenation (particularly matrix free Ca levels) and electron transport capacity for regenerating mitochondrial membrane potential (Av|/m), which in turn depends on cytochrome c content and IMM proton leak. Building upon previous studies demonstrating a role of mitoKATp channel and PKCe in modulating these components, Project 1 will further explore the signal transduction pathways protecting mitochondria from the MPT priming and trigger components under conditions generally relevant to ischemia/reperfusion by integrating functional studies with proteomic analyses. In collaboration with the Heart Biology Core, the functional studies will use spectrofluorometric, imaging (fluorescent, confocal and high voltage electron microscopy), and adenoviral gene transfer techniques to study mitochondria and cardioprotection at three levels: isolated mitochondria, in situ mitochondria in permeabilized myocytes, and isolated myocytes. In collaboration with Project 2, Project 3, the Proteomic Core, and the Heart Biology Core, the proteomic analyses will dissect mitochondrial protein complexes associated with the voltage-dependent anion channel (VDAC, an MPT pore component), PKCe and the novel mitochondrial protein phosphatase PP2CK in protected and unprotected intact hearts. Using this integrated approach, Project 1 will (1) further characterize the mechanisms by which ischemic/reperfusion elements promote the MPT priming and trigger components, and how mitoKATp channel agonists are protective in this setting;(2) define the roles of key mitochondrial subproteomes in protection against the MPT priming and trigger components; (3) characterize Avym depolarization waves induced by anoxia/reoxygenation to define their association with cytochrome c release and MPT and their responsiveness to mitoKATp activation and cardioprotective signaling pathways.